There has been significant progress in the design and synthesis of numerous nucleotide analogues bearing a modified nucleobase moiety or unnatural sugar and that are substrates for polymerases. Modifications at the phosphate moiety are introduced to increase the stability of a nucleotide toward enzymatic degradation or to mask the phosphate negative charge and facilitate its penetration into a cell. Common strategy in nucleotide prodrug design is protecting a phosphate moiety with a labile masking group. Removal of a masking group liberates a nucleoside monophosphate entity to be transformed into a nucleoside triphosphate (hereinafter referred as NTP), a substrate for intracellular enzymes. However, even after removal of the masking group, phosphorylation and activation of nucleoside monophosphate remains a problem due to substrate specificity of cellular kinases. Therefore, design of a nucleotide analogue that would allow bypassing the kinase activation pathway while behaving as a direct polymerase substrate would be a considerable challenge.
Treatment of certain viral infections has always been a challenging task due to ability of some viruses to integrate into a host's genome. Therefore, the viral enzymes that are critical for viral genome replication and integration are regarded as the most effective targets for the design of anti-viral agents.
A lot of attention has been given to studying mechanisms of action of Human Immunodeficiency Virus (type 1) (HIV-1) and developing specific inhibitors towards this very challenging and important target. One of the enzymes that are essential for the HIV replication is HIV reverse transcriptase (HIV RT). The function of this enzyme is to use a viral RNA genome and a reverse transcriptase to synthesize a double stranded DNA for integration into a host genome. Because this step is critical for the propagation of the viral infection, HIV reverse transcriptase (RT) is an excellent target for anti-viral treatment. Currently, two major classes of RT inhibitors (RTIs) exist and are administered for treatment of HIV infection. Non-nucleoside reverse transcriptase inhibitors (NNRTs) are a group of compounds that act through the allosteric inhibition by binding to a hydrophobic site, or a pocket in close proximity to the active site of HIV RT. The other group of RTIs is represented by nucleoside reverse transcriptase inhibitors (NRTIs) that bind directly to the active site and interfere with the polymerization reaction and DNA synthesis.
Nucleoside reverse transcriptase inhibitors are designed to be recognized as substrates for RT and incorporated into a growing strand for further termination of chain elongation. Inhibition of reverse transcriptase activity and chain termination by NRTIs is achieved by introduction of structural modifications to the sugar moiety. The elongation of the DNA strand by a polymerase requires a nucleophilic attack of the 3′-OH group to the a phosphorus atom of an incoming nucleotide. Therefore, nucleoside analogs that lack the 3′-OH group or have it substituted with other functional groups (for instance, N3, F, H) not capable of the nucleophilic attack and formation of phosphodiester bond would act as chain terminators.
Termination of DNA or RNA synthesis with nucleoside analogues is a common and one of the most efficient strategies in the treatment of viral infections, regardless of various side effects and cell toxicity. The therapeutically active form of a nucleoside analogue is a nucleoside triphosphate. However, at the physiological pH nucleoside triphosphates are negatively charged molecules and thus they can not penetrate cellular membranes. Hence, RT inhibitors are usually administered as biologically inactive free nucleosides or as monophosphate prodrugs where a phosphate group is masked with a lipophilic group.
There are three steps of kinase-mediated activation of anti-viral nucleosides. At first, transformation to a monophosphate derivative takes place through the action of a cytoplasmic nucleoside kinase (for instance, thymidine kinase and deoxycytidine kinase). Furthermore, a nucleoside 5′-monophosphate kinase catalyzes the conversion of a nucleoside monophosphate to a nucleoside diphosphate. Finally, a diphosphate derivative is phosphorylated by a nucleoside 5′-diphosphate kinase (NDK) to provide an anti-viral nucleoside analog in its activated (phosphorylated) form. The efficiency of phosphorylation depends on substrate specificity of kinases. For instance, in the case of the AZT phosphorylation cascade, conversion from the nucleoside monophosphate to the nucleoside diphosphate becomes a rate limiting step as thymidylate kinase (TMPK) catalyzes this conversion significantly slower than in the case of the natural substrate (TMP). The consequences of this inefficiency are accumulation of AZTMP in the cytosol and decreased therapeutic concentration of AZTTP, the activated nucleoside form. However, it was determined that high levels of AZTMP have an inhibitory effect on thymidylate kinase by competing with its natural substrate (TMP) and resulting in reduced levels of TDP and TTP. Moreover, increased levels of AZT and its phosphorylated derivatives also affect other enzymes of the de novo dNTPs synthesis resulting in skewed natural nucleotide concentrations.
Therefore, administration of free NRTIs, which often relies on intracellular phosphorylation and activation, has significant drawbacks. One of the possible solutions is a prodrug or pronucleotide approach. In the prodrug approach, the monophosphate moiety is “masked” with a labile functional group which also serves to facilitate passage of a “masked” nucleotide inside the cell. Once inside the cell, a masking group is removed either enzymatically or through chemical activation. Removal of the masking group affords a free nucleoside monophosphate intracellularly where it can be further phosphorylated by TMPK and NDK. Thus, although the prodrug approach facilitates delivery of an inhibitory nucleoside inside the cell and eliminates the need for initial phosphorylation by a nucleoside kinase, phosphorylation by TMPK and NDK are still required.
Besides delivery and bio-distribution challenges, another drawback that is often associated with anti-viral therapy is emergence of resistant strains. In the case of HIV-1, the drug resistance is developed by appearance of mutations that would allow HIV RT to discriminate NRTIs for natural nucleotides or remove an incorporated unnatural nucleobase by excision reactions. It has also been shown for herpes simplex virus (HSV) that reduction in anti-herpetic activity of acyclovir, a drug activated by thymidine kinase phosphorylation and commonly used for treatment of HSV infections, is mostly associated with thymidine kinase dependent resistance. Established strategies to manage acyclovir-resistant HSV infections include administration of anti-viral drugs acting directly on a viral DNA polymerase (foscarnet, cidifovir) or by modulating immune response of a patient. However, the later approach is not always feasible and the former one could worsen patient's condition since these medications impose a significant level of toxicity.
Therefore, considering all aforementioned aspects of therapy directed to inhibit viral polymerases and reverse transcriptases, a nucleotide analogue that would not depend on activation by nucleoside/nucleotide kinases whilst serving as a natural substrate mimic, would be of a great interest. In particular, there is a need in the art for the development of novel phosphate-modified nucleosides that meet the requirements for successful polymerase recognition, including good chelating properties and spatial features to form stable enzyme-substrate complexes, and whereby their incorporation reaction into oligonucleotides is not stalled.